1. Field of the Invention
The present invention relates to a vesicular-arbuscular mycorrhizal fungi inoculum and methods for the production and the utilization thereof for plant growth enhancement.
2. Discussion of the Prior Art
VA (vesicular-arbuscular) mycorrhizal fungi are beneficial fungi in that they infect the feeding roots of plants and stimulate uptake of phosphorus from the soil. Hyphae of the fungus grow outwardly from the root well beyond the phosphate depletion zone (the zone from which the available phosphate has already been consumed by the plant). Selected VA-mycorrhizal fungi have been shown to enhance the growth of numerous plants of economic importance [Jeffries, CRC Critical Reviews in Biotechnology, Vol. 5, "Use of Mycorrhizae in Agriculture," pages 319-357 (1987)], including vegetables [Haas et al, Agron. J., Vol. 79, "Vesicular-arbuscular Mycorrhizal Fungus Infestation and Phosphorus Fertigation to Overcome Pepper Stunting After Methyl Bromide Fumigation," pages 905-910 (1987); Mohandas, Plant and Soil, Vol. 98, "Field Responses to Tomato (Lycopersicon esculentum) to Inoculation With a VA-mycorrhizal Fungus Glomus fasciculatum and Azotobacter virelandii, pages 295-297 (1987); Plenchette et al, Plant and Soil, Vol. 70, "Growth Response of Several Plant Species to Mycorrhizae in a Soil of Moderate Phosphorus Fertility I. Mycorrhizal Dependency Under Field Conditions," pages 197-209 (1983)]; field crops [Baltruschat, Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz, Vol. 94, "Field Inoculation of Maize with Vesicular-arbuscular Mycorrhizal Fungi by Using Expanded Clay as Carrier Material for Mycorrhiza," pages 419-430 (1987); Hall, J. Agr. Sci., Vol. 102, "Field Trials Assessing the Effect in Inoculating Agricultural Soils With Endomycorrhizal Fungi," pages 725-731 (1984); Medina et al, Biol. Fert. Soils, "Growth Response of Field-grown Siratro (Macroptilium atropurpureum Urb.) and Aeschynomene americana L. to Inoculation With Selected Vesicular-arbuscular Mycorrhizal Fungi," Vol. 9, pages 54-60 (1990)] and native plants used for revegetation [Dehgan et al, Bartow, Fla.: Florida Institute of Phosphate Research, "Propagation and Mycorrhizal Inoculation of Indigenous Florida Plants for Phosphate Mine Revegetation, No. 03-053-076, 225 pages (1989); Sylvia, J. Coastal Res., Vol. 5, "Nursery Inoculation of Sea Oats with Vesicular-arbuscular Mycorrhizal Fungi and Outplanting Performace of Florida Beaches," pages 747-754 (1989)]. Nonetheless, VA-mycorrhizal fungi are not used widely in crop production, partially because inoculum sources are limited and application technologies are not well developed.
A major limitation to the utilization of VA-mycorrhizae in crop production is the inability to produce sufficient amounts of fungal inoculum. The VA-mycorrhizal fungi have not been grown successfully in pure culture and are considered obligate symbionts [Hepper, VA Mycorrhiza, Powell and Bagyaraj, eds. (CRC Press, Boca Raton, Fla. (1984))]. Due to this limitation, these fungi are usually maintained and increased in pot cultures [Ferguson et al, "Methods and Principles of Mycorrhizal Research," N. C. Schenck, ed. (American Phytopathological Society, St. Paul, Minn. (1982))], sand [Thompson, Can. J. Bot., Vol. 64, pages 2282-2294 (1986)] or expanded clay [Dehne et al, Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz, Vol. 93, No. 4, pages 415-424 (1986)]. Pot cultures are comprised of host plants, mycorrhizal fungi and soil microflora and microfauna, and are influenced by the physical and chemical properties of the potting medium [Sylvia, "Applications of Mycorrhizal Fungi In Crop Production," J. J. Ferguson, ed. (University of Florida, Gainesville, Fla. (1984))] . It is not surprising that the quality and quantity of propagules produced for inoculum by this method varies widely due to the many interactions among these variables.
Inoculum of VA-mycorrhizal fungi may consist of soil (containing colonized root fragments, spores and hyphae), colonized roots alone or spores alone. The use of soil and root inoculum has limited commercial value because these systems can become easily contaminated by other VA-mycorrhizal fungi and plant pathogens [Schenck et al, Phytopathology, Vol. 72, page 950 (1982)]. Single-species cultures of VA-mycorrhizal fungi that are relatively free of other contaminating organisms are best achieved by the use of spore inoculum.
Several alternatives to the pot-culture system have been proposed for inoculum production. The VA-mycorrhizae have been formed on root-organ cultures [Mosse et al, Physiol. Plant Pathology, Vol. 5, page 215 (1975); Miller-Wideman et al, Can. J. Microbiol., Vol. 30, page 642 (1984)]. However, colonization and sporulation are limited in these monoxenic systems. The VA-mycorrhizae have also been established in solution culture [Howeler et al, New Phytol., Vol. 90, page 229 (1982); Mosse et al, Can. J. Bot., Vol. 62, page 1523 (1984); Elmes et al, Can. J. Bot., Vol. 62, page 1531 (1984); Crush et al, N. Z. J. Agricultural Research, Vol. 24, page 371 (1981); U.S. Pat. No. 4,294,037]. For example, Elmes and Mosse reported approximately 50% colonization of Zea mays roots after nine weeks in a nutrient film system. The inoculum produced in their nutrient system, however, was primarily colonized root material. In general, sporulation in solution culture systems has been poor. This is due to the fact that VA-mycorrhiza are inhibited by excessive moisture and poor aeration in the natural environment.
The VA-mycorrhizae have also been grown in aeroponic culture [Hung et al, Appl. Environ. Microbiol., Vol. 54, "Production of Vesicular-arbuscular Mycorrhizal Fungus Inoculum in Aeroponic Culture," pages 353-357 (1988)].
Various strategies have been proposed to apply inoculum of VA-mycorrhizal fungi in agriculture [Jarstfer et al, Soil Microbial Technologies, B. Metting, ed. (Marcel Dekker, Inc.), "Inoculation Techniques and Inoculum Production of Vesicular-arbuscular Mycorrhizal Fungi" (in press)]. Inocula-containing soil is considered impractical due to its bulk and the risk of contamination; however, chopped roots in peat blocks [Warner, U.S. Pat. No. 4,551,165, "Mycorrhizal Seed Pellets" (1985)] and spores within a porous matrix [Baltruschat, supra] have been proposed for field application. Since the cost of inoculum production is high, a need exists for methods to process inocula of VA-mycorrhizal fungi for efficient distribution.
Colonized roots of a Glomus sp. can serve as effective inoculum due to the presence of intraradical vesicles [Biermann et al, New Phytol., Vol. 95, "Use of Vesicular-arbuscular Mycorrhizal Roots, Intraradical Vesicles and Extraradical Vesicles As Inoculum," pages 97-106 (1983)]. However, for efficient handling inoculum should be processed into small and uniform pieces. Such inoculum can then be pelletized [Crush et al, Endomycorrhizas, Sanders and Mosse, eds. (Academic Press, N.Y.), "Preliminary Results on the Production of Vesicular-arbuscular Mycorrhizal Inoculum by Freeze Drying" (1975)] or used in fluid-drill systems [Bryan, Applications of Mycorrhizal Fungi in Crop Production, J. Ferguson, ed. (University of Florida, Gainesville, Fla.), "Fluid Drilling of Vegetable Crops: A Technique Adaptable for Mycorrhizal Field Inoculation," pages 46-47 (1984)]. Roots colonized by a Glomus sp. have been macerated [Biermann et al, supra] and variously milled [Warner, supra; Jackson et al, Soil Sci. Soc. Amer. Proc., Vol. 36, "Effects of Vesicular-arbuscular Mycorrhizae on Growth and Phosphorous Content of Three Agronomic Crops," pages 64-67 (1972)]. However, milling was found to reduce inoculum potential. Warner, supra, reported nearly a 50% drop in propagules when air-dried peat inoculum was milled to a size of 850 .mu.m.
It is an object of the present invention to provide a novel VA-mycorrhizal inoculum composition which is advantageously applied in agricultural methods for enhancing plant growth and which has a high propagule density.